Methods and compositions for sequencing nucleic acids using charge

ABSTRACT

The invention provides methods and compositions, and systems for determining the identity of nucleic acids in nucleotide sequences, including sequences with one or more homopolymer regions. The methods of the invention include improvements so as to accurately identify sequences, including the difficult homopolymer sequences that are encountered during nucleotide sequencing, such as pyrosequencing.

FIELD OF THE INVENTION

The invention relates to methods and compositions, and systems fordetermining the identity of nucleic acids in nucleotide sequences, andin particular, sequences that contain consecutive repeats of aparticular base.

BACKGROUND OF THE INVENTION

Over the past 30 years, the amount of DNA sequence information that hasbeen generated and deposited into Genbank has grown exponentially. Manyof the next-generation sequencing technologies use a form of sequencingby synthesis (SBS), wherein specially designed nucleotides and DNApolymerases are used to read the sequence of single-stranded DNAtemplates in a controlled manner. Pyrosequencing is a form of SBS whichallows sequencing of a single strand of DNA by synthesizing thecomplementary strand along it, one base pair at a time, and detectingwhich base was actually added at each step.

Rotherberg et al. teach the use of large arrays of chemically sensitiveFETs (chemFETs) or more specifically ISFETs for monitoring reactions,including for example nucleic acid (e.g., DNA) sequencing reactions,based on monitoring analytes present, generated or used during areaction. See U.S. Patent Application Publication No. 20100137143,hereby incorporated by reference. More generally, arrays including largearrays of chemFETs may be employed to detect and measure static and/ordynamic amounts or concentrations of a variety of analytes (e.g.,hydrogen ions, other ions, non-ionic molecules or compounds, etc.).Rotherberg et al. teach the measurement of hydrogen ions, rather thanthe pyrophosphate normally measured in pyrosequencing.

However, there are types of sequences which are difficult to sequence(even with these newer approaches), and in particular, sequences thatcontain consecutive repeats of a particular base. What is needed is animproved method which addresses the ability to sequence all types ofsequence.

SUMMARY OF THE INVENTION

DNA sequences often have so-called homopolymeric regions (e.g.T-T-T-T-T). Pyrosequencing of DNA template containing the homopolymericregions produces results which make it very difficult to identify theexact sequence from the data (e.g. is the region T-T-T-T or T-T-T-T-T?)because pyrosequencing is done with unblocked nucleotides and relies onthe magnitude of the signal to determine the number of incorporationsfor the homopolymeric region. This becomes a very large problem as readlengths increase because secondary effects such as non-specific bindingreactions and synthesis dephasing are cumulative with the number ofincorporation reaction cycles. These effects contribute to themeasurement noise and make it more difficult to use a single detectorintensity value as an accurate indicator of the number of incorporationsin a homopolymeric region. It is also a problem where the sequencecontains multiple regions of this type.

The invention relates to methods and compositions, and systems fordetermining the identity of nucleic acids in nucleotide sequences, andin particular, sequences that contain one or more consecutive repeats ofa particular base (so-called homopolymeric regions). In one embodiment,method for sequencing nucleic acids comprising, a) incorporating one ormore nucleotides into a plurality of nucleic acids in one or morereaction chambers in contact with one or more ion detectors, whereinsaid nucleotides comprise a 3′-OH blocking group, said blocking grouppreventing any further nucleotide incorporation and any furtherextension of the nucleic acids in which the nucleotide is incorporatedunless removed, and b) detecting hydrogen ions released upon nucleotideincorporation by said one or more ion detectors. In one embodiment, saidblocking group is a removable chemical moiety. It is not intended thatthe present invention be limited by the nature of the blocking group. Inone embodiment, said removable chemical moiety comprises a disulfidebond. In one embodiment, said removable chemical moiety comprises anazido group. In one embodiment, said removable chemical moiety comprisesan azidomethyl ether. In one embodiment, said removable chemical moietycomprises an aminoxy group. In one embodiment, said removable chemicalmoiety comprises an oxime group. It is also not intended that thepresent invention be limited to a particular type of sequence with aparticular homopolymer region. In one embodiment, a portion of thesequence of said nucleic acid comprises consecutive identical bases ofthe formula X_(n), where X is any base and n is a whole number between 3(e.g. A-A-A, G-G-G, C-C-C, etc.) and 10. In one embodiment, the nucleicacid to be sequenced is immobilized (e.g. on a bead, in a well, etc.)For example, one may immobilize template DNA on a solid surface by its5′end. Incorporation of the nucleotides typically takes place in aprimer which becomes a complementary extension strand of the strandbeing sequenced. One may accomplish this by annealing a sequencingprimer to the nucleic acid (e.g. to a consensus sequence that has beenintroduced into the nucleic acid to be sequence) and introducing a DNApolymerase (including non-natural polymerases which have been mutated toimprove performance, including incorporation of nucleotide analogs withbulky groups).

While the above-described embodiment utilizes the charge coming from the3′-OH group of an already incorporated nucleotide in the chain (i.e.resulting from the loss of H when the new nucleotide is incorporated),the present invention also contemplates embodiments, where the chargecomes from chemical groups designed into the nucleotide. Suchembodiments allow for leaving groups or cleavable groups with larger(more easily detectable) charges, including both positive and negativecharges. Thus, in another embodiment, the present invention contemplatesa method for sequencing nucleic acids comprising, a) incorporating oneor more nucleotides into a plurality of nucleic acids in one or morereaction chambers in contact with one or more charge detectors(including ion detectors), wherein said nucleotides comprise a cleavablemoiety (or label) and a 3′-OH blocking group, said blocking grouppreventing any further nucleotide incorporation and any furtherextension of the nucleic acids in which the nucleotide is incorporatedunless removed, b) cleaving said cleavable moiety (or label) underconditions such that a charged moiety is produced, and c) detecting saidcharged moiety with said one or more charge detectors. In oneembodiment, said charged moiety is positively charged. In anotherembodiment, said charged moiety is negatively charged. Indeed, one typeof nucleotide (e.g. T) might have a group that can be cleaved so as toproduce a positive charge, while another type of nucleotide (e.g. G)might have a group that can be cleaved so as to produce a negativecharge (thereby allowing for the nature of the charge to correlate withthe nature/identity of the base). In yet another embodiment, saidcharged moiety may have a different magnitude for each type ofnucleotide. For example, one type of nucleotide (e.g. T) might have onelevel of positive charge, while another nucleotide (e.g. A) might havetwo (or three, or four, etc.) times that level of positive charge. Theseembodiments could be combined such that pyrimidines (C, T, U) have apositive charge, but differ in magnitude, while purines (A and G) have anegative charge, but differ in magnitude. On the other hand, thepyrimidines could have the negative charge, but differ in magnitude,which the purines could have the positive charge, but differ inmagnitude. In either case, charge and magnitude of charge would permitidentification of the incorporated base.

In one embodiment, there is a wash step prior to step b) which removesunincorporated nucleotides (and any other reagent). It is sufficientthat this wash steps remove the majority of excess reagents (and morepreferably 90% of such reagents), even if not removing %100. It is notintended that the present invention be limited by the nature of theagent used to cleave the moiety or label. In one embodiment, said labelis cleaved enzymatically. In one embodiment, said label is cleavedchemically. It is also not intended that the present invention belimited to a particular type of sequence with a particular homopolymerregion. In one embodiment, the present invention contemplates a portionof the sequence of said nucleic acid comprises consecutive identicalbases of the formula X_(n), where X is any base and n is a whole numberbetween 3 and 10. In one embodiment, the cleavable label is attachedthrough a cleavable linker to the base of said nucleotide.

Definitions

To facilitate understanding of the invention, a number of terms aredefined below, and others are found elsewhere in the specification.

The term “plurality” means two or more.

The term “nucleotide sequence” refers to a polymer comprisingdeoxyribonucleotides (in DNA) or ribonucleotides (in RNA). Nucleotideshave a base selected from the group of adenine (A), guanine (G),cytosine (C), thymine (T), and uracil (U).

The term “interrogation position” when made in reference to a nucleotidesequence refers to a location of interest in the sequence, such as thelocation at which the identity of a nucleic acid is sought to bedetermined.

The terms “cleavable moiety, ” “cleavable marker,” and “cleavable label”are interchangeably used to describe a chemical moiety that, whenattached to a composition of interest (e.g. to the base of anucleotide), acts as a marker for the presence of the composition ofinterest. The “label” need not be detectable visually (although suchembodiments are also contemplated since some dyes have charge). Thelabel is preferably detected by charge (e.g. after cleavage). Thepresent invention envisions labels that would carry a net negative orpositive charge. For example, one can use mono, di and tricarboxylicacids (acetic, oxalic, malonic, succinic, citric etc., since they willbe deprotonated) attached via a cleavable linker for negative charge(e.g. the cleavable linker attached to the base or another part of thenucleotide). Or one could use mono, di or triamines (since they will beprotonated) for positive charge. Finally, one can use labels that wouldrelease protons upon cleavage.

The invention's compositions and methods contemplate using modifiednucleotides. The terms “nucleotide” and “nucleic acid” refer toconstituents of nucleic acids (DNA and RNA) that contain a purine orpyrimide base, such as adenine (A), guanine (G), cytosine (C), uracil(U), or thymine (T)), covalently linked to a sugar, such as D-ribose (inRNA) or D-2-deoxyribose (in DNA), with the addition of from one to threephosphate groups that are linked in series to each other and linked tothe sugar. The term “nucleotide” includes native nucleotides andmodified nucleotides.

“Native nucleotide” refers to a nucleotide occurring in nature, such asin the DNA and RNA of cells. In contrast, “modified nucleotide” refersto a nucleotide that has been modified by man, such as using chemicaland/or molecular biological techniques compared to the nativenucleotide. The terms also include nucleotide analogs attached to one ormore probes to facilitate the determination of the incorporation of thecorresponding nucleotide into the nucleotide sequence. In oneembodiment, nucleotide analogues are synthesized by linking a uniquelabel through a cleavable linker to the nucleotide base or an analogueof the nucleotide base, such as to the 5-position of the pyrimidines (T,C and U) and to the 7-position of the purines (G and A), to use a smallcleavable chemical moiety to cap the 3′-OH group of the deoxyribose orribose to make it nonreactive, and to incorporate the nucleotideanalogues into the growing nucleotide sequence strand as terminators,such as reversible terminators and irreversible terminators. Detectionof the unique label (e.g. by charge) will yield the sequence identity ofthe nucleotide. Upon removing the label and the 3′-OH capping group, thepolymerase reaction will proceed to incorporate the next nucleotideanalogue and detect the next base. Other nucleotide analogs that containmarkers, particularly cleavable markers, are also contemplated, such asthose configured using allyl groups, azido groups, and the like, andwhich are further described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows synthesis of 3′-O-azidomethyl-dNTPs where the steps denotetreatment with (i) DMSO, AcOH, Ac₂O, 48 h; (ii) SO₂Cl₂, dry CH₂Cl₂, 1-2h; (iii) NaN₃ in DMF, 3 h; (iv) NH₄F in MeOH, 16-20 h; (v) (MeO)₃PO,POCl₃ then (t-Bu₃NH)₄P₂O₇, TEAB, 1 h; vi) NH₄OH.

FIG. 2 shows synthesis of 3′-O-azidomethyl-dGTP where the steps denotetreatment with (i) DMSO, AcOH, Ac₂O, 48 h; (ii) Ph₂NCOCl, DIEA, Pyridine3 h; (iii) SO₂Cl₂, dry CH₂Cl₂, 1-2 h; (iii) NaN₃ in DMF, 3 h; (iv) NH₄Fin MeOH, 24 h; (v) (MeO)₃PO, POCl₃ then (t-Bu₃NH)₃P₂O₇H, TEAB, 1 h; (vi)NH₄OH.

FIG. 3 shows exemplary nucleotide structures with 3′-OH group protectionthat can be cleaved by mild oxidation reactions.

GENERAL DESCRIPTION OF THE INVENTION

The present invention, in one embodiment, contemplates using the blockednucleotides described herein together with large scale FET arrays formeasuring one or more analytes (e.g. ions and charged moieties). In thevarious embodiments disclosed herein, FET arrays include multiple“chemFETs,” or chemically-sensitive field-effect transistors, that actas chemical sensors. An ISFET is a particular type of chemFET that isconfigured for ion detection, and ISFETs may be employed in variousembodiments disclosed herein. Other types of chemFETs contemplated bythe present disclosure include ENFETs, which are configured for sensingof specific enzymes. It should be appreciated, however, that the presentdisclosure is not limited to ISFETs and ENFETs, but more generallyrelates to any FET that is configured for some type of chemicalsensitivity.

According to yet other embodiments, the present disclosure is directedgenerally to inventive methods and apparatus relating to the delivery tothe above-described large scale chemFET arrays of appropriate chemicalsamples to evoke corresponding responses. The chemical samples maycomprise (liquid) analyte samples in small reaction volumes, tofacilitate high speed, high-density determination of chemical (e.g., ionor other constituent) concentration or other measurements on theanalyte.

For example, some embodiments are directed to a “very large scale”two-dimensional chemFET sensor array (e.g., greater than 256 k sensors),in which one or more chemFET-containing elements or “pixels”constituting the sensors of such an array are configured to monitor oneor more independent chemical reactions or events occurring in proximityto the pixels of the array. In some exemplary implementations, the arraymay be coupled to one or more microfluidics structures that form one ormore reaction chambers, or “wells” or “microwells,” over individualsensors or groups of sensors of the array, and apparatus which deliversanalyte samples to the wells and removes them from the wells betweenmeasurements. Even when microwells are not employed, the sensor arraymay be coupled to one or more microfluidics structures for the deliveryof one or more analytes to the pixels and for removal of analyte(s)between measurements.

In various embodiments, an analyte of particular interest is hydrogenions, and large scale ISFET arrays according to the present disclosureare specifically configured to measure pH. In other embodiments, chemFETarrays may be specifically configured to measure pH or one or more otheranalytes that provide relevant information relating to a particularchemical process of interest. In various aspects, the chemFET arrays arefabricated using conventional CMOS processing technologies, and areparticularly configured to facilitate the rapid acquisition of data fromthe entire array (scanning all of the pixels to obtain correspondingpixel output signals). See U.S. Patent Application Publication No.20090026082, hereby incorporated by reference.

DESCRIPTION OF PREFERRED EMBODIMENTS

In one embodiment, the nucleotide analogs are exemplified by nucleotidecompositions comprising compounds of the following general structure:

Where PG1 stands for protective group that is selectively removable and,and CL stands for cleavable linker, which is also selectively cleavable,and R is selected from the group of H, OH, F, NH₂. Several particularembodiments of this invention are contemplated. In one embodiment thesenucleotide compositions can be incorporated into the nucleic acid bynucleic acids modifying enzymes in a controlled fashion to decode theidentity of the bases encoded by the marker moiety M. Once the markermoiety has been cleaved off, identity of the base may be decoded bymeasuring the change in charge in the reaction chamber due to thereleased marker moieties. In one embodiment, this invention contemplatesthe use of the cleavable linkers based on the “trimethyl lock” mechanismor the “1,6-rearrangement” mechanism. The 3′-O-protective groups whichact as reversible terminators can also be cleaved off to enable additionof the next nucleotide. This invention contemplates the use ofazidomethyl, methylaminoxy, disulfide, aminoxy, oxime and allyl groupsas reversible 3′-OH terminators.

Methods for synthesizing exemplary nucleotide analogs that containcleavable markers configured using azido groups are shown in FIGS. 1 and2.

The invention contemplates the use of the cleavable linkers based on the“trimethyl lock” mechanism or the “1,6-rearrangement” mechanism. The3′-O-protective groups which act as reversible terminators can also becleaved off to enable addition of the next nucleotide. The inventioncontemplates the use of azidomethyl, aminooxy, methylaminoxy and ally!groups as reversible 3′-OH terminators.

A. Cleavable Linkers (Cl)

Cleavable linkers are exemplified by trimethyl lock based linkers and1,6-rearrangement linkers as further described below.

1. Trimethyl Lock Based Linkers

Cleavable linkers are the linkers linking the marker molecule M to thebase and these can be selectively cleaved using specific cleavingagents. Specifically, this invention contemplates the use of a“trimethyl lock” structure as the cleavage mechanism. These structuresare well known in the chemical arts and have been used before incontrolled drug release applications. The general structures ofcleavable trimethyl lock based linker utilized in particular embodimentsof the present invention are shown below:

The above shows exemplary embodiment A where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker, and X is a divalentgroup selected from NH, O, S.

The above shows exemplary embodiment B where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker, and X is NH.

The above shows exemplary embodiment C where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker, and X is a divalentgroup selected from NH, O, S, and Y is a selectively removableprotective group.

The above shows exemplary embodiment D where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker, X is NH, and Y is anazidomethyl group.

The linkers in the present invention leverage the ability of thetrimethyl lock system to create cleavably linked nucleotides.

2. 1,6-Rearrangement Linkers

The invention contemplates another category of cleavable linkers linkingthe detectable marker moiety to the nucleotide that are based on 1,6quinone methide rearrangement mechanism (Carl et al. (1981). J. Med.Chem. 24(5):479-480; Duimstra et al. (2005). J. Am. Chem. Soc. 127(37):12847-12855). These structures are well known in the chemical arts andthey have been used before for the controlled drug release applicationsand for chemical synthesis (Azoulay et al. (2006) Bioorganic & MedicinalChemistry Letters 16(12): 3147-3149; Murata et al. (2006) TetrahedronLetters 47(13): 2147-2150). The general structures of cleavable 1,6rearrangement mechanism based linker utilized in some embodiments of thepresent invention are shown below:

The above shows exemplary embodiment E, where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker and Y is a selectivelyremovable protective group.

The above shows exemplary embodiment F, where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker.

The above shows exemplary embodiment G where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker, and X is a divalentgroup selected from the following: NH, O, S.

The above shows exemplary embodiment where BASE is selected from anyribo- or deoxyribo-nucleobases: adenosine, cytidine, guanosine,thymidine and analogs, M is a detectable marker, and X is a divalentgroup selected from the following: NH, O, S. The cleavage is driven hereby the reducing agent and nucleophilic attack of the resulting aminogroup on the carbonyl followed by cyclization. This mechanism has beenused before for the development of protective groups for applications inthe carbohydrate and nucleoside chemistry (Wada et al. (2001).Tetrahedron Letters 42(6): 1069-1072; Xu et al. (2002) CarbohydrateResearch 337(2): 87-91).

The cleavable linker attachment to the base moiety can be achieved invariety of ways that are well known in the art. Among these is the useof linkers based on 1) propargylamino nucleosides, 2) aminoallylnucleosides, and 3) propargylhydroxy nucleosides.

B. Protective Groups (PG1)

The invention contemplates nucleotide compositions comprising thefollowing blocking or protective groups (PG1) that reside on the 3′-OHgroups of the nucleotides: 1) 3′-O-Azidomethyl ethers, 2)3′-O-disulfide, 3) 3′-O-methylaminoxy, 4) 3′-aminoxy, 5) 3′-oxime and 6)3′-O-allyl.

With respect to the 3′-O-Azidomethyl ethers, exemplary protective groupsthat reside on the 3′-OH groups of the nucleotides that are within thescope of this invention are 3′-O-azidomethyl groups. These groups can beremoved using mild reducing agents, such as tri(carboethoxy)phosphine(TCEP).

With respect to the 3′-O-disulfide group, the 3′-O-disulfide group canbe removed under mild oxidative conditions, for example using in usingmild reducing agents, such as .tri(carboethoxy)phosphine (TCEP)

With respect to the 3′-O-methylaminoxy, 3′-aminoxy, and 3′-oxime groups,they can be removed under mild oxidative conditions, for example usingin situ generated nitrous acid (such as from sodium nitrite).

As to the 3′-O-allyl group, this protective group can be removed using avariety of reducing agents, including transition metal complexes (Pd,Rh).

Examples of PG1 protective groups are shown in FIG. 3.

Experimental

The following examples serve to illustrate certain exemplary embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

Materials And Methods

The following is a brief description of the exemplary materials andmethods used in the following Examples. All solvents and reagents werereagent grades, purchased commercially and used without furtherpurification. Protected nucleosides5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine,N⁴-benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine,N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine,N²-isobutyryl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine werepurchased from CNH Technologies, Inc. All other chemicals were purchasedfrom Sigma-Aldrich.

EXAMPLE 1 Synthesis Of 3′-O-Azidomethyl Nucleotides

The synthesis of 3′-O-azidomethyl-dNPTs is described in FIG. 1. Briefly,reaction of 5′-O-TBDMS-2′-deoxynucleosides (5) with a mixture of DMSO,acetic acid, and acetic anhydride installed the 3′-O-methylthiomethylgroup (3′-O-MTM, 6), which upon treatment with SO₂Cl₂ converted toactivated 3′-O—CH₂Cl (7). The latter can be monitored in TLC as 3′-OH(5) after dissolving in wet organic solvent due to fast hydrolysis ofthe —CH₂Cl group. The 3′-O—CH₂Cl-2′-deoxynucleoside (7) is then treatedwith NaN₃ in dry DMF without purification to convert to 3′-O—CH₂N₃ (8).3′-O-azidomethyl-2′-deoxynucleosides of A,T, and C (9a-9c) were obtainedin good yield after deprotection of the 5′-O-TBDMS group as described inthe FIG. 1. Similar synthesis route for guanosine(G, 9d), lead only verylow yield (>10%) due to formation of a number of side reaction products.To circumvent this, a new method was introduced for the synthesis ofguanosine analog (14) which is described in the FIG. 2, which involvedprotection of the O⁶— group by diphenycarbamoyl group. After protectionof this particular group, the intermediate (12-14) became less polar,making easier to purify, and lead good overall yield in the azidomethylgroup installation step.

EXAMPLE 2 Synthesis of N⁶-benzoyl-3′-O-(azidomethyl)-dA (9a)

The following describes exemplary synthesis steps for compounds shown inFIG. 1.

A. Synthesis ofN⁶-Benzoyl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxyadenosine(6a)

3.0 g N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine (5a)(6.38 mmol) was dissolved in a mixture consisting of 11.96 mL DMSO, 5.46mL acetic acid, and 17.55 mL acetic anhydride and stirred at roomtemperature for 48 h. The reaction mixture was then neutralized treatingwith a sufficient amount of saturated NaHCO₃ solution and extracted withCH₂Cl₂ (3×100 mL). The combined organic extract was then washed with asaturated NaHCO₃ solution (100 mL), dried over Na₂SO₄, and concentratedunder vacuum. The resultant yellowish oil was then purified on silicagel column (Hex: EtOAc/1:1 to 1:4) to obtain the productN⁶-benzoyl-3′-O-(methylthiomethyl)-5′-O-(tent-butyldimethylsilyl)-2’-deoxyadenosine(6a) as white powder in 71% yield (2.4 g, R_(f) 0.6, EtOAc: hex/7:3).HR-MS: obs. m/z 530.2273, calcd. for C₂₅H₃₆O₄N₅SiS 530.2257 [M+H]⁺.¹H-NMR (CDCl₃): δ_(H) 9.00 (s, 1H), 8.83 (s, 1H), 8.35 (s, 1H), 8.05 (d,J=7.6 Hz, 2H), 7.62 (m, 1H), 7.55 (m, 2H), 6.55 (t, J=7.19 Hz, 1H), 4.73(m, 2H), 4.68 (m, 1H), 4.24 (m, 1H), 3.88 (dd, J =11.19, 3.19 Hz, 1H),2.74-2.66 (m, 2H), 2.35 (s, 3H), 0.94 (s, 9H) and 0.13 (s, 6H) ppm.

B. Synthesis of N⁶-benzoyl-3′-O-(azidomethyl)-2′-deoxyadenosine (9a)

To 0.4 gN⁶-benzoyl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxyadenosine(0.76 mmol) dissolved in 7 mL dry CH₂Cl₂ was treated with 0.4 mLcyclohexene and 155 μL SO₂Cl₂ (1.91 mmol) at 0° C. for 2 h. During thistime the starting material completely converted to 7a which was shown bydisappearance of the starting material and appearance of 3′-OH analog(5a) in TLC (EtOAC:Hex/7:3, R_(f)˜0.3; the 3-CH₂Cl (7a) could notdetected in TLC due to decomposition in TLC plate to 5a). Then solventwas removed by rotary evaporation and kept about 10 minutes in highvacuum pump. Then dissolved in 5 mL dry DMF and treated with 400 mg NaN₃(6.6 mmol) at room temperature for 3 h. Then the reaction mixture waspartitioned in H₂O/CH₂Cl₂, the combined organic part was dried overNa₂SO₄ and concentrated by rotary evaporation. The crude sample was thendissolved in 5 mL MeOH and treated with 300 mg NH₄F (8.1 mmol) more than38 h. Then MeOH was removed by rotary evaporation. After partioning inH₂O/EtOAc, the combined organic part was dried over Na₂SO₄,concentrated, and purified by silica gel column chromatography (100%EtOAc to 98:2, EtOAc/MeOH) resulting 150 mg of 9a as white powder (48%yield in three steps). HR-MS: Obs. m/z 411.1530, calcd for C₁₈H₁₉O₄N₈411.1529 [M+H]⁺. ¹H-NMR (CDC₃): δ_(H) 8.84 (brs, 1H), 8.70 (brs, 1H),8.08 (m, 1H), 7.76-7.54 (m, 5H), 6.47 (t, J=5.6 Hz, 1H), 4.83 (m, 2H),4.78 (m, 1H), 4.39 (m, 1H), 4.09 (d, J=12.78 Hz, H₅′, 1H), 3.88 (d,J=12.78 Hz, H₅″, 1H), 3.09 (m, H₂′, 1H), and 2.65 (m, H₂″, ¹H) ppm.

EXAMPLE 3 Synthesis of 3′-O-azidomethyl-dT (9b)

The following describes exemplary synthesis steps for compounds shown inFIGS. 1.

A. Preparation of3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine(6b)

2.0 g 5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine (5b) (5.6 mmol)was dissolved in a mixture consisting of 10.5 mL DMSO, 4.8 mL aceticacid, and 15.4 mL acetic anhydride and stirred for 48 h at roomtemperature. The mixture was then quenched by treating with a saturatedNaHCO₃ solution and extracted with EtOAc (3×100 mL). The combinedorganic extract was then washed with a saturated solution of NaHCO₃ anddried over Na₂SO₄, concentrated under vacuum, and finally purified bysilica gel column chromatography (Hex: EtOAc/7:3 to 1:1). The3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine(6b) was obtained as white powder in 75% yield (1.75 g, R_(f)=0.6, hex:EtOAc/1:1). HR-MS: Obs. m/z 417.1890, cald. for C₁₈H₃₃N₂O₅SSi 417.1879[M+H]⁺. ¹H-NMR (CDCl₃): δ_(H) 8.16; (s, 1H), 7.48 (s, 1H), 6.28 (m, 1H),4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H), 3.78-3.90 (m, 2H), 2.39 (m,1H), 2.14, 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s, 3H), 0.93 (s, 9H), and0.13 (s, 3H) ppm.

B. Preparation of 3′-O-(azidomethyl)-2′-deoxythymidine (9b)

To 1.095 g3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxythymidine(6b) (2.6 mmol) dissolved in 10 mL dry CH₂Cl₂ were added 1.33 mLcyclohexene and 284 μL SO₂Cl₂ (3.5 mmol) at 0° C. and stirred at theice-cold temperature for 1.5 h. Then the flask temperature was broughtto room temperature and transferred to a round bottom flask. Thevolatiles were removed by rotary evaporation followed by high vacuumpump. Then the crude sample was dissolved in 5 mL dry DMF and 926 mgNaN₃ (15.4 mmol) was added to it and stirred for 3 h at roomtemperature. The crude sample was dispersed in 50 mL distilled water andextracted with CH₂Cl₂ (3×50 mL), the organic extracts were combined anddried over Na₂SO₄ and concentrated by rotary evaporation. The crudesample was then dissolved in MeOH (5 mL) and treated with NH₄F (600 mg,16.2 mmol) for 24 h at room temperature. Then reaction mixture wasconcentrated and partitioned between H₂O/CH₂Cl₂ and the combined organicextract was dried over Na₂SO₄, concentrated, and purified the product bysilica gel column chromatography using Hex: EtOAc/1:1 to 2:5 resultingthe final product (9b) as white powders (˜550 mg, 71% yield in threesteps, R_(f)=0.3, Hex: EtOAc/1: 1.5). HR-MS: Observed m/z 298.1146,calcd for C₁₁H₁₆O₅N₅ 298.1151 [M+H]⁺. ¹H-NMR (CDC₃): δ_(H) 8.30 (brs,1H), 7.40 (s, 1H), 6.14 (t, J=6.8 Hz, 1H), 4.79-4.70 (m, 2H), 4.50 (m,1H), 4.16 (m, 1H), 4.01-3.84 (m, 2H), 2.45 (m, 2H) and 1.95 (s, 3H) ppm.

EXAMPLE 4 Synthesis of N⁴-Benzoyl-3′-O-(azidomethyl)-dC (9c)

The following describes exemplary synthesis steps for compounds shown inFIG. 1.

A. Preparation ofN⁴-Benzoyl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine(6c)

3.5 g N⁴-benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (5c)(7.65 mmol) was dissolved in a mixture consisting of 14.7 mL DMSO, 6.7mL acetic acid, and 21.59 mL acetic anhydride and stirred for 48 h atroom temperature. During this period of time, a complete conversion toproduct was observed by TLC (R_(f)=0.4, EtOAc:hex/10:1). The mixture wasthen neutralized with a saturated NaHCO₃ solution and extracted withCH₂Cl₂ (3×100 mL). The combined organic extract was then washed withsaturated solution of NaHCO₃ and dried over Na₂SO₄, and concentratedunder vacuum. The product was then purified by silica gel columnchromatography (EtOAc: hex/2:1 to 9:1) to obtainN⁴-benzoyl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine(6c) as white powder in 73% yield (2.9 g, R_(f)=0.6, EtOAc:hex/9:1).HR-MS: obs. m/z 506.2134, cald. for C₂₄H₃₆O₅N₃SiS [M+H]⁺. 506.2145.¹H-NMR (CDCl₃): δ_(H) 8.43 (d, J=7.1 Hz, 1H), 7.93 (m, 2H), 7.64 (m,1H), 7.54 (m, 3H), 6.30 (m, 1H), 4.62 & 4.70 (2×d, J=11.59 Hz, 2H), 4.50(m, 1H), 4.19 (m, 1H), 3.84 & 3.99 (2×dd, J=11.59 & 2.79 Hz, 2H), 2.72(m, 1H), 2.21 (m, 1H), 2.14 (s, 3H), 0.99 (s, 9H), and 0.16 (s, 6H) ppm.

B. Preparation of N⁴-Benzoyl-3′-O-(azidomethyl)-2′-deoxycytidine (9c).To 0.5580 gN⁴-benzoyl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine(6c)

(1.04 mmol) dissolved in 8 mL dry CH₂Cl₂ were added 0.56 mL cyclohaxeneand 220 μL SO₂Cl₂ (2.7 mmol) at 0° C. and stirred at the ice-coldtemperature for 1 h. During this time, the starting material convertedto the chlorinated product as shown by the 3′-OH (5c) compound in theTLC. The volatiles were then removed under vacuum and resuspended in dryDMF (5 mL) and treated with NaN₃ (400 mg, 6.6 mmol) and stirred for 2 hat room temperature. The sample was then partitioned between water andCH₂Cl₂ and the organic extracts were combined and dried over Na₂SO₄ andconcentrated under vacuum. The crude sample was then dissolved in MeOH(5 mL) and treated with NH₄F (600 mg, 16.2 mmol) for 20 h at roomtemperature. Then solvent was removed under vacuum and extracted withCH₂Cl₂ and the organic extract was then dried over Na₂SO₄ andconcentrated under vacuum. The sample was then purified by silica gelcolumn chromatography (Hex:EtOAc 1:4 to 1:10), and the product (9c) wasobtained as white powdery substance (˜200 mg, 50% yield in three steps,R_(f)=0.5, EtOAc:Hex/5: 0.5). HR-MS: Obs. m/z 387.1408, calcd forC₁₇H₁₉O₅N₆ 387.1417 [M+H]⁺. ¹H-NMR (CDC₃): δ_(H) 8.30 (d, J=7.2 Hz, 1H),7.93 (d, J=7.50 Hz, 1H), 7.66-7.51 (m, 5H), 6.18 (t, J=6.4 Hz, 1H),4.81-4.68 (m, 2H), 4.52 (m, 1H), 4.25 (m, 1H), 4.08-3.88 (m, 2H), 2.69(m, 1H), and 2.50 (m, 2H) ppm.

EXAMPLE 5 Synthesis ofN²-isobutyryl-O⁶-diphenylcarbamoyl-3′-O-azidomethyl-dG (14)

The following describes exemplary synthesis steps for compounds shown inFIG. 2.

A. Preparation ofN²-isobutyryl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(11)

5 g of N²-isobutyryl-5′-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(11.0 mmol) dissolved in 21 mL dry DMSO was treated with 10 mL aceticacid and 32 mL acetic anhydride, and stirred for 48 h at roomtemperature. The crude reaction mixture was then neutralized by adding aK₂CO₃ solution, and extracted with ethyl acetate (100×3 mL). Thecombined organic extract was then washed with saturated NaHCO₃ solution,dried over Na₂SO₄ and concentrated under vacuum. Then reaction mixturewas purified by a silica gel column chromatography resulting the product11 as white powder (3.9 g, 69% yield; R_(f)=0.35, CH₂Cl₂:MeOH/20:1).HR-MS: Obs. m/z 512.2344 cald. for C₂₂H₃₈O₅N₅SiS 512.2363 [M+H]⁺. ¹H-NMR(CDCl₃): δ_(H) 12.0 (s, 1H), 8.95 (brs, 1H), 8.09 (s, 1H), 6.24 (t,J=6.8 Hz, 1H), 4.73 (m, 2H), 4.66 (m, 1H), 4.16 (m, 1H), 3.81 (m, 2H),2.76 (m, 1H), 2.59 (m, 1H), 2.54 (m, 1H), 2.21 (s, 3H), 1.29 (m, 6H),0.91 (s, 9H), and 0.10 (s, 6H) ppm.

B. Synthesis ofN²-isobutyryl-O⁶-diphenylcarbamoyl-3′-O-(methylthiomethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(12)

To 1.0 gN²-isobutyryl-3′-O-(methylthimethyl)-5′-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(11, 1.95 mmol) dissolved in 22 mL dry pyridine were addeddiphenylcarbamoyl chloride (0.677 g, 2.92 mmol) and 1.02 mLN,N-diisopropylethylamine, and stirred at room temperature for 3 h undernitrogen atmosphere. The reaction mixture became dark red during thistime. The solvent was removed under high vacuum, and product was thenpurified by silica gel column chromatography using EtOAc:hex/1:1 to 7:3as mobile phase. The product 12 was isolated as yellowish powder (1.09g, ˜80% yield; R_(f)=0.7, EtOAc:hex (1:1)). HR-MS: Obs. m/z 707.3068calcd. for C₃₅H₄₇O₆N₆SiS 707.3047 [M+H]⁺. ¹H-NMR (CDCl₃): δ_(H) 8.25 (s,1H), 7.94 (brs, 1H), 7.47-7.37 (m, 10H), 6.42 (m, 1H), 4.75 (m, 2H),4.71 (m, 1H), 4.18 (m, 1H), 3.88-3.70 (m, 2H), 2.80 (m, 1H), 2.60 (m,1H), 2.19 (s, 3H), 1.30 (d, J=7.2 Hz, 6H), 0.93 (s, 9H) and 0.14 (s, 6H)ppm.

C. Preparation ofN²-isobutyryl-O⁶-diphenylcarbamoyl-3′-O-azidomethyl-2′-deoxyguanosine(14)

To 786 mg 12 (1.1 mmol) dissolved in 8 mL dry CH₂Cl₂ was treated with0.56 mL cyclohexene and 180 μL SO₂Cl₂ (2.2 mmol) at 0° C. and stirredfor 1.5 h at the same temperature. The solvent was then removed byrotary evaporation, and further dried under high vacuum for 10 minutes.The crude product was then dissolved in 5 mL dry DMF and reacted with600 mg NaN₃ (10 mmol) at 0° C. and stirred at room temperature for 3 h.Reaction mixture was then partitioned H₂O/CH₂Cl₂, the combined organicextract was then dried over Na₂SO₄, and concentrated by rotaryevaporation. The crude was then dissolved in 5 mL dry MeOH, treated with500 mg NH₄F (13.5 mmol) at room temperature for more than 24 h. ThenMeOH solvent was removed by rotary evaporation, and partitioned(H₂O/CH₂Cl₂). The combined organic part was dried over Na₂SO₄ andconcentrated by rotary evaporation and purified by silica gel columnchromatography resulting pure product of 14 as white powder (230 mg,˜36% yield in three steps; hex: EtOAc 1:1 to 1:5, (R_(f)=˜0.3,Hex:EtOAc/1:4). HR-MS: Obs. m/z 588.2343, calcd for C₂₈H₃₀O₆N₉ 588.2319[M+H]⁺. ¹H-NMR (DFM-d₆): δ_(H) 8.64 (brs, 1H), 7.48-7.34 (m, 10H), 6.36(t, J=7.0 Hz), 4.93 (m, 2H), 4.76 (m, 1H), 4.04 (m, 1H), 3.57 (m, 1H),3.34 (m, 2H), 2.97 (m, 1H), 2.81 (m, 1H), and 1.10 (m, 6H).

EXAMPLE 6 General Method For The Preparation Of 3′-O-Azidomethyl-Dntps

The protected 3′-O-azidomethyl nucleoside (0.3 mmol) and proton sponge(75.8 mg; 0.35 mmol) were dried in a vacuum desiccator over P₂O₅overnight before dissolving in trimethyl phosphate (0.60 mL). Thenfreshly distilled POCl₃ (33 μL, 0.35 mmol) was added drop-wise at 0° C.and the mixture was stirred at 0° C. for 2 h. Subsequently, awell-vortexed mixture of tributylammonium pyrophosphate (552 mg) andtributylamine (0.55 mL; 2.31 mmol) in anhydrous DMF (2.33 mL) was addedin one potion at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M, 15 mL, pH 8.0) was thenadded and the mixture was stirred for 1 h at room temperature. Then 15mL of NH₄OH was added and stirred overnight at room temperature. Theresulting mixture was concentrated in vacuo and the residue was dilutedwith 5 mL of water. The crude mixture was then purified with anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M). Further purification by RP HPLC to givecorresponding target as colorless syrup:

EXAMPLE 7 3′-O-Azidomethyl Nucleotides Cleavage

The 3′-O-azidomethyl group cleavage can be accomplished with a varietyof reducing agents such as phosphines. The cleavage agents that areparticularly desirable are those that are soluble in aqueous media anddo not cause any damage to the DNA. One particularly desirable agent istri(carboethoxy)phosphine (TCEP).

The 3′-O-azidomethyl nucleotides can be separated from nativenucleotides using RP HPLC. In the next experiment, the kinetics of the3′-O-azidomethyl TTP cleavage was studied. For this purpose, a 1 mMsolution of nucleotide was prepared in water and mixed with 50mMsolution of TCEP/400 mM of Tris at pH 8.0 and incubated at 55 deg C. forvarious periods of time. After the incubation, the reaction was stoppedby mixing with 4 M NaOAc at pH=4.3 and an aliquot of reaction mixture(0.5 nmole of nucleotide) was injected and separated on the RP HPLCcolumn. The integrated peak area was then plotted against time.

We claim:
 1. A method for sequencing nucleic acids comprising, a)incorporating one or more nucleotides into a plurality of nucleic acidsin one or more reaction chambers in contact with one or more iondetectors, wherein said nucleotides comprise a 3′-OH blocking group,said blocking group preventing any further nucleotide incorporation andany further extension of the nucleic acids in which the nucleotide isincorporated unless removed, and b) detecting hydrogen ions releasedupon nucleotide incorporation by said one or more ion detectors.
 2. Themethod of claim 1, wherein said blocking group is a removable chemicalmoiety.
 3. The method of claim 2, wherein said removable chemical moietycomprises a disulfide bond.
 4. The method of claim 2, wherein saidremovable chemical moiety comprises an azido group.
 5. The method ofclaim 4, wherein said removable chemical moiety comprises an azidomethylether.
 6. The method of claim 2, wherein said removable chemical moietycomprises an aminoxy group.
 7. The method of claim 2, wherein saidremovable chemical moiety comprises an oxime group.
 8. The method ofclaim 1, wherein a portion of the sequence of said nucleic acidcomprises consecutive identical bases of the formula X_(n), where X isany base and n is a whole number between 3 and
 10. 9. A method forsequencing nucleic acids comprising, a) incorporating one or morenucleotides into a plurality of nucleic acids in one or more reactionchambers in contact with one or more charge detectors, wherein saidnucleotides comprise a cleavable label and a 3′-OH blocking group, saidblocking group preventing any further nucleotide incorporation and anyfurther extension of the nucleic acids in which the nucleotide isincorporated unless removed, b) cleaving said cleavable label underconditions such that a charged moiety is produced, and c) detecting saidcharged moiety with said one or more charge detectors.
 10. The method ofclaim 9, wherein said charged moiety is positively charged.
 11. Themethod of claim 9, wherein said charged moiety is negatively charged.12. The method of claim 9, wherein there is a wash step prior to step b)which removes unincorporated nucleotides.
 13. The method of claim 9,wherein said label is cleaved enzymatically.
 14. The method of claim 9,wherein said label is cleaved chemically.
 15. The method of claim 9,wherein a portion of the sequence of said nucleic acid comprisesconsecutive identical bases of the formula X_(n), where X is any baseand n is a whole number between 3 and
 10. 16. The method of claim 9,wherein the cleavable label is attached through a cleavable linker tothe base of said nucleotide.